Optimizing Heterologous Production of CRISPR-AsCas12a Protein in Escherichia coli

doi:10.21203/rs.3.rs-4535821/v1

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Optimizing Heterologous Production of CRISPR-AsCas12a Protein in Escherichia coli

https://doi.org/10.21203/rs.3.rs-4535821/v1

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The CRISPR-Cas12a system is a groundbreaking tool that has seen an ample use for genome editing and diagnostics in biotechnology and biomedicine research labs. Despite its increasing use, there is a lack of studies on optimizing Cas12a protein production at lab-scale using straightforward protocols. This study aimed on enhancing the lab-scale recombinant production of Acidaminococcus sp Cas12a protein (AsCas12a) in E. coli. Through careful adjustments of simple parameters, the production of AsCas12a was remarkably increased. Optimized conditions involved using the BL21(DE3) strain, TB medium with 1% glucose, induction with 0.3 mM IPTG for at least 6–9 h and incubation at 30°C. Notably, these conditions deviate from conventional production protocols for Cas12a and related proteins such as Cas9 from Streptococcus pyogenes. Upon combination of all optimized conditions bacterial production of AsCas12a improved ~ 3 times, passing from 0.95 mg / mL of bacterial lysate volume, for non-optimized conditions, to 3.73 mg/mL in the optimal ones. The production yield of AsCas12a protein, after chromatographical purification increased ~ 4.5 times, from 5.2 to 23.4 mg/L (culture volume) without compromising its functionality at all. The purified AsCas12a protein retained full activity for programmable in vitro DNA cis-cleavage and for collateral trans-activity, which was used to detect the N gene from SARS-CoV-2. This optimized method offers an efficient and high-yield AsCas12a protein production using materials and conditions that are accessible to many research labs around the world.

CRISPR-Cas12a

Protein biosynthesis

Recombinant production

Optimization

Lab-scale production

Lab-scale production of Cas12a was highly boosted using a simple strategy.
Optimized conditions increased AsCas12a production yield up to 4.5 times.
Method offers efficient, straightforward and high-yield Cas12a production.

The CRISPR-Cas12a system, formerly known as Cpf1, stands as a potent molecular tool with diverse applications in genome editing, molecular diagnostics, and various biotechnological and biomedical fields (Paul and Montoya 2020; Shi et al. 2021; Khan and Sallard 2023). Given the manifold applications of the CRISPR-Cas12a system, there exists a widespread interest in the small-scale, high-yield biosynthesis of the Cas12a protein among research laboratories in both developed and non-developed countries (Bandyopadhyay et al. 2020; Jirawannaporn et al. 2022; Han et al. 2022; Paenkaew et al. 2023). Many laboratories around the world need a continuous supply of the protein to carry on and continue with their research activities. Although Cas12a protein is available commercially, its high cost makes research labs to prefer to produce and purify themselves the protein. However, the current available lab protocols are low efficiency and have not been optimized. Despite the increasing application of Cas12a, there remains a noticeable lack of studies focused on its small lab-scale production without the need to use a bioreactor or specialized bacterial strains. While some groups have reported the recombinant production of Francisella novicida Cas12a protein (Zetsche et al. 2015; Dong et al. 2016; Strohkendl et al. 2018a; Chen et al. 2018), only very few reports (Mohanraju et al. 2018; Martin et al. 2023) have described detailed protocols for the biosynthesis of other relevant orthologues such as Acidaminococcus sp Cas12a protein (AsCas12a). Surprisingly, there is a notable gap in the literature regarding the systematic study of production of Cas12a, underscoring the need for comprehensive investigations in this area. Consequently, an exploration of the recombinant production of Cas12a proteins is imperative to advance CRISPR-Cas12a-based research, with a particular focus on the AsCas12a variant in this study.

The endonuclease AsCas12a is classified as a type V CRISPR-Cas system, as it serves as the sole effector protein responsible for DNA cleavage. Cas12a possesses key attributes that position it as a robust alternative to the widely utilized CRISPR-Cas9 system (Swarts and Jinek 2018). Notably, the crRNA of Cas12a is shorter than Cas9 gRNA (40–44 vs 100 nt), and it cleaves dsDNA in a staggered manner. Cas12a exhibits a higher precision in binding to dsDNA sequences compared to Cas9, boasting a substantial affinity (K_D ~fM) and displays a pronounced trans-cleavage activity upon ssDNA (commonly referred to as collateral activity) (Jinek et al. 2012; Zetsche et al. 2015; Dong et al. 2016; Gao et al. 2016). These distinctive features make the Cas12a protein an excellent choice within the CRISPR-Cas systems for both fundamental and applied research.

AsCas12a forms a ribonucleoprotein complex (RNP) in conjunction with a CRISPR RNA (crRNA, also known as guide RNA or gRNA). The RNP makes a highly specific cleavage of both DNA strands upon binding the target DNA with a remarkable affinity (K_D of 54 fM) (Zetsche et al. 2015; Strohkendl et al. 2018a). Particularly effective with T-rich genomes, AsCas12a recognizes the PAM 5’ TTTV 3’ (Kim et al. 2017). Comprising 1,307 amino acid residues and a molecular weight of 151.2 kDa, the AsCas12a is structured into REC and NUC lobes (Dong et al. 2016; Gao et al. 2016). The NUC lobe contains the RuvC-I, II and III domains, responsible for the ultimate cleavage of dsDNA. Distinct from SpyCas9, AsCas12a exhibits unique features (Swarts and Jinek 2018). AsCas12a uses a shorter guide RNA, lacks a tracrRNA, cleaves dsDNA with 5’ overhangs and has a longer distance between the seed sequence and the cleavage site (Swarts and Jinek 2018). Despite these differences, AsCas12a robustly and efficiently cleaves dsDNA in human cells (Zetsche et al. 2015), displaying on-target efficiencies comparable to the widely used SpyCas9 (Kleinstiver et al. 2016). All this underscores the need of studies focused on optimizing the recombinant production of AsCas12a in E. coli, the most commonly employed heterologous expression system of choice for many research labs around the world (Rosano and Ceccarelli 2014).

This study presents a systematic study aimed to identify optimal conditions for the lab-scale production of recombinant AsCas12a in E. coli as expression system, comparing them with initial conditions based on standard available protocols (Mohanraju et al. 2018; Martin et al. 2023) (Fig. 1). It was studied the effect the influence of several parameters, including E. coli strain, IPTG concentration, culture media, media supplementation with glucose, and post-induction temperature (Fig. 1A). The protein expression was monitored under various conditions, and the most effective combinations were identified to ensure efficient protein expression. The significance of studies that report improved conditions to express and purify CRISPR-Cas proteins is of large relevance, as they contribute to advancing the entire CRISPR-Cas field by facilitating the availability of proteins for experimentation. It was considered that the optimized conditions for the heterologous production of AsCas12a protein in E. coli will prove beneficial to research laboratories engaged in CRISPR-based studies that have available basic equipment and materials for heterologous bacterial expression.

Plasmids

The pMBP-AsCas12a plasmid was employed for the production of AsCas12a protein (Fig. 1), generously provided by Prof. Ilya Finkelstein at the University of Texas-Austin (Addgene #113430) (Fig. 1B). This plasmid harbors an ampicillin resistance gene and the AsCas12a gene, featuring a His10-tag, all under the control of a T7 promoter. The plasmid pPET28a-SARS-CoV-2-geneN was utilized for cis and trans cleavage assays of the AsCas12a protein, a generous gift from Dr. Luis Brieba de Castro from LANGEBIO-UGA, Mexico, containing the N gene from the SARS-CoV-2 Wuhan reference genome.

Materials

The Luria Bertani (LB) Broth culture medium was purchased from Invitrogen (Waltham, Massachusetts, USA). The highly enriched Terrific Broth (TB) medium was prepared with 24 g/L yeast extract (Formedium, Hunstanton, UK), 12 g/L Tryptone (Sigma-Aldrich, St. Louis, Missouri, USA), 4 mL glycerol (JT Baker, Phillipsburg, NJ, USA), and 0.17 M phosphate buffer (pH 7) (K₂HPO₄ and KH₂PO₄, Sigma-Aldrich). Glucose, ampicillin, kanamycin, 2-Mercaptoethanol and bromophenol blue were purchased from Sigma-Aldrich. Isopropil ß-D-1-thiogalactopyranoside (IPTG) was obtained from Invitrogen (Carlsbad, USA). Agar, acrylamide, bis-acrylamide, sodium dodecyl sulfate (SDS), ammonium persulfate, and glycine were purchased from Biorad (Hercules, California, United States). N, N, N′, N′-Tetramethyl ethylenediamine (TEMED) was acquired from IBI Scientific (Dubuque, Iowa, United States), while Tris was obtained from Gold Bio (St. Louis, Missouri, United States). Furthermore, gRNAs and ssDNA reporter probe were synthesized by Integrated DNA Technologies-IDT (Coralville, USA).

Bacterial strains and culture media preparation

The strains employed in the assays for recombinant protein biosynthesis, BL21 (DE3), Rosetta (DE3), BL21(pLysS), Tuner^™ (DE3), and NiCo (DE3), were generously provided by Dr. Patricia Cano Sánchez and Dr. Corina-Diana Ceapă from the Institute of Chemistry, UNAM, Mexico. The LB broth was prepared by dissolving 12.5 g of LB media powder in 1 L of distilled water. TB solution was prepared as mentioned above. These media solutions were sterilized using an autoclave (Sterilite 24, Fisher Scientific, Waltham, USA). As needed, LB and TB media were enriched by supplementing with glucose, reaching a final concentration of 1% (w/v), previously sterilized using 0.2 µm filters (Sartorius, Germany).

Bacterial transformation and plasmid production

Bacterial transformation was achieved through the thermal shock method. Competent E. coli DH5α (or any other strain) were placed on ice for 5 min with 2 µL plasmid. The mixture underwent a temperature shift to 42°C for 30 s, followed by an incubation on ice for 5 min. The sample then was mixed with 100 µL LB and incubated at 37°C for 1 h. The mixture was added to agar plates supplemented with 100 µg/mL ampicillin and incubated overnight at 37°C. A colony of bacteria transformed with the plasmid was grown overnight in 5 mL LB medium, incorporating the necessary antibiotic (ampicillin or kanamycin) at 100 µg/mL. Subsequently, plasmids were purified from the culture using the ZymoPURE Plasmid Miniprep Kit (Zymo Research, California, USA).

Expression of protein AsCas12a

The initial experimental conditions were based on previously published protocols for the expression of Cas12a (Mohanraju et al. 2018; Strohkendl et al. 2018a) (Fig. 1C). To initiate the pre-inoculum, a colony was selected from an agar plate and cultivated in a 250 mL Erlenmeyer flask containing 100 mL media (LB or TB, depending on the assay) supplemented with 100 µg/mL of ampicillin. The pre-inoculum was incubated at 37°C for 16 h and 225 rpm using a MaxQ 4,000 shaker incubator equipped with controlled temperature settings (Thermo Scientific, Waltham, Massachusetts). Following this, the pre-inoculum was diluted to an optical density (OD) of 0.1 in 100 mL of culture media (TB or LB, with or without 1% glucose, incorporating 100 µg/mL of ampicillin). The culture was then incubated at 37°C with 225 rpm until the OD reached a value of 0.6–0.8 (approximately 1.5 h). OD was measured in a BioPhotometer plus spectrophotometer (Eppendorf, Hamburg, Germany). Then, the culture was incubated at the desired experimental temperature (12, 18, 30 or 37°C) with 225 rpm. After 30 min, IPTG induction was carried out (0.3, 0.6, 1, 1.3, 1.6 mM), and the culture was incubated for the designated time.

Cell lysis and sample preparation

Bacterial samples taken during the growth curves were treated in the following way before their analysis of protein expression. 1 mL of bacterial culture was centrifuged at 4°C for 5 min at 13,000 rpm using a microcentrifuge AccuSpin Micro 17R (Fisher Scientific, Waltham, USA). The supernatant was discarded, and the pellet was resuspended in 100 µL cell lysis buffer [200 mM 2-Mercaptoethanol, 25% glycerol, 10% SDS, 0.5 M Tris-HCl (pH 6.8), and 0.5% bromophenol blue] and heated up to 95°C for 5 min using an Isotemp Thermoblock (Fisher Scientific, Waltham, USA). Finally, the solution was centrifuged at 4°C for 5 min at 13,000 rpm and the supernatant was recovered and stored at -20°C for further analysis, then only the soluble fraction was further analyzed. Cell lysis buffer without bromophenol blue was used when total protein concentration present in the bacteria culture was measured by Nanodrop. When very dense cultures were lysed, they were centrifugated during 55 min to eliminate the excess of debris.

Quantification of total soluble protein

The total protein quantification was performed using a NanoDrop One UV-Vis spectrophotometer (Thermo Scientific, Waltham, Massachusetts) and by micro-bicinchoninic acid assay (BCA) using the Pierce™ BCA Protein Assay Kit (ThermoScientific). For NanoDrop, approximately 1.5 µL of the cell lysate were scanned using the protein A280 program after a buffer baseline correction determined by the device. For micro–BCA microplate Assay 150 µL of sample (2 µL of bacterial lysate diluted to a volume of 500 µL) were loaded into a 96-well microplate by triplicate together with the albumin standard (prepared following the instructions of the protocol). Then, 150 µL of the BCA working reagent was added to each well and the mixture was incubated for 2h at 37°C to finally read in a Cytation 5 Multi Mode Reader (BioTek, Santa Clara, USA) at 562 nm. The obtained results were analyzed and plotted using GraphPad 10.

SDS-PAGE analysis

Protein expression from lysed samples was analyzed by electrophoresis in SDS-PAGE gel using a 247.6 mM Tris-glycine (pH 8) as running buffer at 160 V for 45 min. Precision Plus Protein™ Kaleidoscope™ Prestained Protein Standard was used as a molecular weight marker (Bio Rad, Hercules, California, United States). Gels were water-washed three times and then stained with SimplyBlue™ Safe Stain (Thermo Fisher, Waltham, USA). Gels were imaged in an Azure 200 Gel Imager (Azure Biosystems, California, USA). Densitometric analysis was carried out using ImageJ software. The percentage of AsCas12a protein present in each sample was determined by measuring the intensity of the band corresponding to the AsCas12a protein band relative to the total protein band intensity present in the lane (Supplementary Fig. S1).

Chromatography protein purification

Protein purification was performed following a previously reported purification protocol (Morales-Moreno et al. 2023). The lysed product from 1 L was purified in an ӒKTA pure™ chromatography system using affinity, ionic exchange, and molecular size exclusion chromatography. Metal affinity chromatography was done with a HisTrapTM HP 5 mL column, and the protein was eluted with 250 mM imidazole at a flow rate of 2 mL/min The Histidine tag was cleaved off using TEV protease during an overnight dialysis at 4°C. The ionic exchange chromatography was carried out using a HiTrap Heparin HP 5 mL column and the protein was eluted with IEx-B buffer (2M potassium chloride) at a flow rate of 2 mL/min. Finally, the molecular size exclusion chromatography was performed using a HiLoad 16/600 Superdex 200 pg 125 mL column and at a buffer flow rate of 0.5 mL/min.

Cis-cleavage activity on pDNA

AsCas12a:crRNA ribonucleoprotein complexes (RNPs) were assembled by incubating for 30 min at 37°C a 1:1 molar mixture of the purified AsCas12a protein and crRNA designed towards the N gene (Morales-Moreno et al. 2023) of SARS-CoV-2 in 1X NEB 2.1 buffer (New England Biolabs, Ipswich, Massachusetts, USA). Then, RNP complexes were mixed with the target dsDNA (pPET28a-SARS-CoV-2-geneN) to a final concentration of 33 nM and 3.3 nM for RNP and pDNA, respectively (molar proportion RNP:pDNA of 10:1) and incubated in a thermoblock at 37°C during 1 h. Then, samples were mixed with Gel Loading Dye 6X (Invitrogen) and evaluated in an 1% agarose gel (Thermo Fisher) using TAE 1X as running buffer (Thermo Fisher) at 140 V for 45 min after staining with SYBR™ Safe DNA Gel Stain (Invitrogen). Gels were imaged with an Azure 300 gel imager system (Azure Biosystems, Dublin, CA).

Trans-cleavage activity on ssDNA

Trans-cleavage activity assay was evaluated following a previous reported protocol (Morales-Moreno et al. 2023). In short, RNPs were assembled as previously indicated and mixed with the target pPET28a-SARS-CoV-2-geneN to a final concentration of 33 nM and 3.3 nM for RNP and pDNA, respectively in 1X NEB 2.1 buffer. As a reporter probe 250 nM of ssDNA attached to FAM and a quencher was used. Trans-cleavage activity on ssDNA was followed by fluorescence at 37°C during 1 h using a Cytation 5 Cell Imaging Multi Mode Reader (BioTek, Santa Clara, USA).

Statistical evaluations

Ordinary one-way ANOVA tests were conducted using the GraphPad Prism 10 software on all the SDS-PAGE densitometry assays results of the supernatants obtained from the lysed bacteria of the cultures (IPTG concentration, Temperature, Culture Media, Glucose supplementation and E. coli Strain) to compare if there was a significant difference between the original parameters and the new experimental parameters. Values of P greater than 0.5 were considered as non-significant.

Effect of IPTG concentration

First, it was explored a 0.3 to 1.6 mM of IPTG concentrations over a 24 h induction period (Fig. 2). The level of AsCas12a protein production upon IPTG concentration was followed by analyzing cell lysates in SDS-PAGE. The SDS-PAGE illustrated successful induction of AsCas12a at all tested IPTG levels (Fig. 2a, Supplementary Fig. S2). Densitometric analysis revealed a decreasing trend in the total AsCas12a protein band percentage relative to the total band intensity with increasing IPTG concentrations (21.1, 19.9, 18.9, 20.3, and 17.1% for 0.3, 0.6, 1, 1.3, and 1.6 mM IPTG, respectively) (Fig. 2b). However, the differences were not statistically significant between them.

Effect of temperature on AsCas12a expression

To assess the impact of temperature on protein expression, recombinant E. coli cultures induced with 0.3 mM IPTG were incubated at various temperatures. After 9 h of induction, cell lysates were analyzed by SDS-PAGE to observe the percentage of produced AsCas12a protein (Fig. 3, Supplementary Fig. S3). AsCas12a protein expression was observed across all temperatures, albeit in varying amounts (Fig. 3a). Densitometric analysis revealed that the highest production of AsCas12a proteins was approximately 27% (of the total soluble protein) at 30°C (Fig. 3b). This contrasts with 12.2% (12°C), 17.9% (18°C), and 13.7% (37°C). The percentage of protein at 30°C represents a ~ 2.2-fold and ~ 1.5-fold increase compared to 12°C and 18°C, respectively.

Effect of culture media

We investigated the impact of highly accessible media, such as LB and TB, for AsCas12a production, and assessed if supplementing them with 1% glucose enhanced production, as seen in SpyCas9 (Carmignotto and Azzoni 2019). After inducing AsCas12a expression with 0.3 mM IPTG and incubating 30°C for 9 h, the percentage of AsCas12a in the total soluble protein (present in cell lysate) was measured with SDS-PAGE (Fig. 4, Supplementary Fig. S4). After comparing LB and TB, it was observed a ~ 1.6-fold improvement (16.9 and 27.2%, respectively). However, supplementing TB with 1% glucose increased AsCas12a protein production from 27.2 to 31.5%, whereas LB + 1% glucose reduced from 16.9 to 15.9% (Fig. 4a). Changing LB to TB + 1% glucose represented a ~ 1.9-fold increment. Notably, TB + 1% glucose production was high enough that the lysed sample caused streaks on SDS-PAGE bands.

Effect of strain

Employing five commonly used E. coli DE3 strains (BL21, Tuner, Rosetta, PlysS, and NiCo), we aimed to identify the most efficient one for AsCas12a biosynthesis under our previously optimized conditions (TB + 1% glucose, 0.3 mM IPTG, and 30°C). After analyzing cell lysates with SDS-PAGE, it was revealed distinct protein production levels among the strains (Fig. 5, Supplementary Fig. S5). BL21 exhibited prominent production, followed by Rosetta and NiCo, while pLysS and Tuner showed the lowest (Fig. 5a). Anomalous electrophoretic mobility was observed in bands with high protein expression, notably in BL21 and Rosetta. The AsCas12a protein production was quantified (Fig. 5b). The quantitative analysis confirmed BL21 as the top performer, 31.5% from the total soluble protein corresponded to AsCas12a protein, followed by Rosetta with 26.7%, NiCo 18.2%, Tuner 15.5%, and pLysS with 14.0%.

Comparison between initial and improved conditions

Next, it was assessed with detail the impact of optimized conditions (BL21(DE3) strain, TB + 1% glucose, 0.3 mM IPTG, and 30°C) and compared against initial non-optimized conditions (BL21(DE3) strain, LB, 1 mM IPTG, and 12°C). This was done by following over 24 h (post-IPTG induction) cell growth kinetics, production of total soluble protein, and production of AsCas12a protein in both conditions (Fig. 6).

After 9h, optimized conditions exhibited significantly faster bacterial growth, reaching a maximum OD of 6.6, whereas non-optimized conditions exhibited an OD of 2.1 absorbance units. It was only after 23h that the non-optimized conditions reached an OD of 2.7 (Fig. 6a). A similar trend was observed for amount of total soluble protein (determined by BCA). After ~ 9h the optimized conditions reached ~ 15 mg/mL (relative to volume of bacterial lysate), whereas for non-optimized conditions was ~ 10 mg/mL (Fig. 6b). Even after 24 h, protein production in non-optimized conditions was still lower (~ 11.5 mg/mL).

To correlate the previous results of production of total soluble protein with AsCas12a protein production, SDS-PAGE and densitometric analysis were carried out (Fig. 6C-E, Supplementary Fig. S6). The quantitative analysis of SDS-PAGE (Fig. 6c) confirmed that ~ 25% of the total soluble protein produced at 9 h corresponded to AsCas12a protein, remaining constant thereafter (Fig. 6d). In contrast, non-optimized conditions showed a slower increase, reaching ~ 13% of AsCas12a protein at 9h and ~ 16% only until 24 h. All this means that the concentration of AsCas12a protein produced at 9 h for optimized conditions was ~ 3.5 mg/mL (relative to volume of cell lysate), and ~ 1.2 mg/mL for non-optimized conditions, representing a ~ 3-fold increment (Fig. 6e). A summary of all values is presented in Table 1.

Table 1

Summary of the results obtained between optimized and non-optimized conditions in bacterial lysates.
	Optical Density (A.U.)^a		Total Soluble Protein (mg/mL)^b		AsCas12a (%)^c		AsCas12a (mg/mL)^d
Time	9 h	24 h	9 h	24 h	9 h	24 h	9 h	24 h
Non-optimized conditions	2.1	2.6	11	11.2	13.2	16	1.2	1.8
Optimized conditions	6.6	7.1	15	15	24.8	23.5	3.1	3.5

^aBacterial growth measured as optical density.

^bTotal amount of cleared soluble protein per volume of bacterial lysate determined by BCA.

^cPercentage of protein AsCas12a in total soluble protein determined by densitometry in SDS-PAGE.

^dTotal amount of protein AsCas12a produced in the bacterial lysate determined from SDS-PAGE and BCA.

Purification and functional evaluation of AsCas12a protein

To assess the functionality of the recombinant AsCas12a produced under optimized conditions, first the protein AsCas12a was purified and then we examined its cis- and trans-cleavage activities (Fig. 7).

The protein was expressed under optimized and non-optimized conditions in 1 L flasks, purified with Ni-NTA affinity, ionic exchange, and molecular size exclusion chromatography (Morales-Moreno et al. 2023), and characterized by SDS-PAGE (Fig. 7a). After purification the production yield of AsCas12a protein was 23.4 mg/L (of bacterial culture), whereas for non-optimized conditions was 5.2 mg/L. Western blot confirmed its identity and successful purification (Supplementary Fig. S7).

Then, it was tested the functional performance of the AsCas12a protein to cleave dsDNA, cis-activity, (Fig. 7b). The cis-cleavage activity of RNP complex (AsCas12a: crRNA) efficiently digested the supercoiled target pDNA (7,253 kbp), while leaving intact the non-target pDNA (9,210 kbp). Finally, it was evaluated the trans-cleavage activity on ssDNA, also called collateral activity (Fig. 7c). It was used two gRNAs that recognize the sequence of the nucleocapsid gene of SARS-CoV-2 located in a pDNA. To evaluate the functional performance of AsCas12a protein, it was followed the fluorescence increase upon cleavage of a FAM-quencher labeled reporter ssDNA after recognition of the target pDNA sequence. The correct trans-cleavage activity on ssDNA was demonstrated by a rapid and sustained increase in fluorescence over time.

CRISPR-Cas systems have become one of the most relevant biomolecular systems for current biotechnological research in many labs around the world. Warrantying a continuous and cheap supply is enormously important for doing basic research and technology development in a productive and timely manner. The system CRISPR-Cas12a is the second most used just below CRISR-Cas9 (Paul and Montoya 2020). Besides genetic engineering, Cas12a has ample applications in biosensor development (Shi et al. 2021; Hernandez-Garcia et al. 2022). In particular, AsCas12a is one of the most used between the various homologous of Cas12a. AsCas12a protein is commonly produced in Escherichia coli; however, an efficient production of the protein has not been reported. Having an optimized production of protein AsCas12a at lab scale would benefit many research labs that continuously use it. Furthermore, optimal production of protein AsCas12a has more chances of being adopted if is based on materials that are easily accessible and available to most research labs, also, if the optimization is based on simple parameters. That is why here we investigated the effect of multiple simple parameters that are important for the efficient production of AsCas12a protein.

First, we explored the effect of 0.3 to 1.6 mM of IPTG concentration over a 24 h induction period (Fig. 2). Our results demonstrated that IPTG concentrations above 0.3 mM had not significant impact. This suggests that 0.3 mM concentration is enough to effectively induce AsCas12a expression in E. coli. This IPTG concentration is very close to previously reported values of 0.13 and 0.2 mM IPTG (Jinek et al. 2012; Mohanraju et al. 2018; Chen et al. 2018; Martin et al. 2023); but contrasts with the 1 mM used by others (Strohkendl et al. 2018b).

Next, we investigated the effect of temperature on AsCas12a expression. After assessing the impact of various temperatures (12, 18, 30 and 37ºC) on protein expression in recombinant E. coli cultures induced with 0.3 mM IPTG (Fig. 3), we found that 30°C was the best post-induction temperature for successful enhancement of protein production. Producing at 30°C increased ~ 2.2-fold and ~ 1.5-fold the production of AsCas12a protein compared to 12°C and 18°C, respectively. This finding is significant since most of the protocols reported for producing AsCas12a using E. coli commonly used temperatures of 12 or 18ºC but not 30ºC (Jinek et al. 2012; Mohanraju et al. 2018; Chen et al. 2018; Martin et al. 2023).

Other aspect of interest in our study was to evaluate the effect of culture media. Analyzing effect of culture media is crucial for optimizing recombinant protein expression (Rosano and Ceccarelli 2014). For that reason, we investigated the impact of highly available media, such as LB and TB, for AsCas12a production (Fig. 4). Also, we assessed if supplementing them with 1% glucose enhanced production, as seen in SpyCas9 (Carmignotto and Azzoni 2019). TB supplemented with 1% glucose resulted advantageous for recombinant production of protein AsCas12a, even surpassing LB, LB + 1% glucose, and TB alone in protein production. The impact on production of adding glucose to TB was very relevant. This positive effect may stem from the role of glucose as an alternative carbon source to an already rich carbon source media (TB contains glycerol), accelerating total biomass and protein production (Choi et al. 2006). Glycerol in TB also aids in preventing acetate formation and serves as an alternative carbon source. Conversely, the negative effect of glucose in LB may be attributed to its transformation into acetates, hindering growth, inhibiting protein formation, and diverting carbon away from biomass to protein product (Wong et al. 2008).

The final parameter was the bacterial strain. The choice of E. coli strain may significantly influence the recombinant production of protein AsCas12a since it has been observed that E. coli strains play important roles for heterologous protein production (Zhang et al. 2022; Pouresmaeil and Azizi-Dargahlou 2023). Among all the studied strains, BL21(DE3) resulted the most efficient (Fig. 5). This is positive judging that is a very common strain in many biotechnological research labs. On the other hand, it could be interesting to explore new E. coli strains that are being developed for efficient heterologous production of proteins under the strategies of synthetic biology (Zarzhitsky et al. 2020; Yang et al. 2022; Zhang et al. 2022).

Finally, we compared between initial and improved conditions. We assessed in detail the impact of combining our best-found conditions against initial non-optimized conditions which were based of typical reported protocols for Cas12a protein production (Martin et al., 2023; Mohanraju et al., 2018; Strohkendl et al., 2018a). The higher values obtained for OD and concentration of total soluble protein and total AsCas12a protein can tell us that our optimization strategy was successful (Fig. 6). Of particular relevance is the production of AsCas12a. The concentration of AsCas12a protein produced at 9 h for optimized conditions was ~ 3.5 mg/mL (relative to volume of cell lysate), which contrast with ~ 1.2 mg/mL obtained with non-optimized conditions. The difference represents a ~ 3-fold increment (Fig. 6e). The striking differences between both conditions could be explained by the usage of the rich media TB + 1% glucose and a higher incubation temperature for the optimized conditions. The efficiency of optimized conditions became evident, indicating their suitability for high-level AsCas12a protein production. In fact, the non-optimized conditions will require much longer than 24 h to produce similar levels of protein. All this demonstrates that our optimized conditions are producing more protein in shorter time.

Verifying the functional performance of the produced and purified AsCas12a protein is of outmost importance. We could verify the cleavage of dsDNA (Fig. 7b), cis-activity, which one of the earliest and most common application of AsCas12a (Zetsche et al. 2015; Kleinstiver et al. 2016; Bandyopadhyay et al. 2020; Han et al. 2022; Yang et al. 2023). Also, the evaluation of the trans-cleavage activity on ssDNA, also called collateral activity, is crucial since it is the base of multiple methods for genetic detection and clinical diagnostics in labs around the world (Chen et al. 2018; Lee Yu et al. 2021; Jirawannaporn et al. 2022; Morales-Moreno et al. 2023; Yang et al. 2023; Paenkaew et al. 2023). The trans-cleavage activity on ssDNA was demonstrated by a rapid and sustained increase in fluorescence over time (Fig. 7c), showcasing the protein full collateral activity and ability to detect specific DNA sequences. Both activities demonstrated to work between acceptable standards.

In conclusion, this study identified several parameters that significantly enhanced the heterologous lab-scale production of AsCas12a protein in recombinant E. coli cultures. Optimized conditions were found for all the studied parameters (IPTG concentration, induction temperature, culture media, and type of bacterial strain). Combination of all optimized conditions increased ~ 3 times the bacterial production of protein AsCas12a in a shorter time (~ 9h). The protein production yield after purification was 23.4 mg/L of culture volume, representing a 4.5-fold increase in comparison to the non-optimized conditions. Importantly, the purified AsCas12a protein retained full cis and trans cleavage activities, which are essential for various downstream CRISPR-Cas applications. These optimized protein production conditions offer a valuable resource for research labs seeking higher yields of functional AsCas12a protein, all in a more efficient timeframe and using a simple protocol that requires accessible materials and equipment.

Funding: This work was supported by UNAM-PAPIIT (IN210121 & IV200820). The authors gratefully acknowledge support from Agencia Mexicana de Cooperacion Internacional para el Desarrollo (AMEXCID)—Secretaria de Relaciones Exteriores Mexico (Proyectos COVID-19).

Conflicts of interest/Competing interests: The authors declare no conflict of interest for this manuscript.

Data availability (data transparency): Supplementary information is included.

Code availability: Not applicable.

Authors' contributions

OSQG and AHG conceived and designed research. OSQG, MDMM, EGVG and RECG conducted experiments. OSQG and AHG analyzed data. AHG wrote the manuscript with input from OSQG. All authors read and approved the manuscript.

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Editorial decision: Minor Revision
10 Jul, 2024
Reviewers agreed at journal
26 Jun, 2024
Reviewers invited by journal
24 Jun, 2024
Editor assigned by journal
15 Jun, 2024
First submitted to journal
05 Jun, 2024

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Optimizing Heterologous Production of CRISPR-AsCas12a Protein in Escherichia coli

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Version 1

Abstract

Figures

Key Points

Introduction

Materials and Methods

Plasmids

Materials

Bacterial strains and culture media preparation

Bacterial transformation and plasmid production

Expression of protein AsCas12a

Cell lysis and sample preparation

Quantification of total soluble protein

SDS-PAGE analysis

Chromatography protein purification

Cis-cleavage activity on pDNA

Trans-cleavage activity on ssDNA

Statistical evaluations

Results

Effect of IPTG concentration

Effect of temperature on AsCas12a expression

Effect of culture media

Effect of strain

Comparison between initial and improved conditions

Purification and functional evaluation of AsCas12a protein

Discussion

Declarations

References

Supplementary Files

Status:

Version 1